Role of ATM and the Damage Response Mediator Proteins 53BP1 and MDC1 in the Maintenance of G₂/M Checkpoint Arrest

Abstract

ATM-dependent initiation of the radiation-induced G 2 /M checkpoint arrest is well established. Recent results have shown that the majority of DNA double-strand breaks (DSBs) in G 2 phase are repaired by DNA nonhomologous end joining (NHEJ), while ϳ15% of DSBs are slowly repaired by homologous recombination. Here, we evaluate how the G 2 /M checkpoint is maintained in irradiated G 2 cells, in light of our current understanding of G 2 phase DSB repair. We show that ATM-dependent resection at a subset of DSBs leads to ATR-dependent Chk1 activation. ATR-Seckel syndrome cells, which fail to efficiently activate Chk1, and small interfering RNA (siRNA) Chk1-treated cells show premature mitotic entry. Thus, Chk1 significantly contributes to maintaining checkpoint arrest. Second, sustained ATM signaling to Chk2 contributes, particularly when NHEJ is impaired by XLF deficiency. We also show that cells lacking the mediator proteins 53BP1 and MDC1 initially arrest following radiation doses greater than 3 Gy but are subsequently released prematurely. Thus, 53BP1؊/؊ and MDC1 ؊/؊ cells manifest a checkpoint defect at high doses. This failure to maintain arrest is due to diminished Chk1 activation and a decreased ability to sustain ATM-Chk2 signaling. The combined repair and checkpoint defects conferred by 53BP1 and MDC1 deficiency act synergistically to enhance chromosome breakage.

Src Family Kinases Promote Silencing of ATR-Chk1 Signaling in Termination of DNA Damage Checkpoint

“…These results suggest that the maintenance of G 2 DNA damage checkpoint arrest is mediated by ATR-Chk1 signaling, which is consistent with previous studies (12,14,16). Exposure to BI2536 strongly inhibited Gö6976-induced mitotic entry and dephosphorylation of Cdk1-Tyr 15 ( Fig.…”

“…A constitutively active mutant of Lyn with the lipid modification (Lyn⌬C) was localized in the nucleus as well as the cell membrane and the perinuclear cytoplasm (Fig. 8C), which is consistent with our previous reports (16,50). By contrast, NLS-Lyn⌬C accumulated in the nucleus.…”

“…Chk1 activity once ATM-dependent DSB end resection occurs and ATR is activated (16). Third, SFKs regulate the ATR-mediated Chk1 phosphorylation that is induced by replication stress in an ATM-independent manner (Figs.…”

“…Because previous works showed that G 2 checkpoint maintenance is largely dependent on Chk1 (12)(13)(14)(15)(16), the possibility that SFK activity is required for the silencing of ATR-Chk1 signaling was examined. At 16 h after the end of Adriamycin exposure, ATM-mediated Chk2 phosphorylation was drastically decreased, but ATR-mediated Chk1 phosphorylation was increased ( Fig.…”

“…Despite the similarity in substrate specificity between ATM and ATR, it is believed that Chk1 is a direct substrate of ATR but not ATM and that Chk2 functions exclusively downstream of ATM in vivo (4). Chk1 activation is essential for the maintenance of G 2 checkpoint arrest in response to DSB induction, and inhibition of Chk1 activity during G 2 checkpoint arrest induces premature mitotic entry even though DNA repair has not been completed (12)(13)(14)(15)(16). Rad17 is another phosphorylation substrate of ATR, and the phosphorylation of Rad17 is required for its interaction with Claspin and Chk1 activation (17)(18)(19).…”